Unlock instant, AI-driven research and patent intelligence for your innovation.

Peptide nucleic acid probe (PNA), kit and method for detection of aspergillus fumigatus and applications thereof

a technology of aspergillus fumigatus and nucleic acid probes, applied in the field of peptide nucleic acid probes, kit and method for detection of aspergillus fumigatus and applications thereof, can solve the problems of lack of methodology standardization, high mortality rate, and conidia can lead to disease, and achieve faster, more robust and specific analysis, prompt and simple

Inactive Publication Date: 2016-05-19
UNIVERSITY OF MINHO +2
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a probe that can recognize a specific gene of microorganisms. This probe has special properties that make it faster, stronger, and more specific than using DNA. The invention also includes blocking probes that can prevent unwanted sequences from interfering with the analysis.

Problems solved by technology

Depending on the host immune system, the inhaled conidia can lead to disease.
The complexity of all the mechanisms involved and the great resistance capacity of A. fumigatus to antifungal substances makes IA hard to cure leading to high mortality rates.
PCR based molecular techniques have been applied as a good alternative but lack of methodology standardization and the possibility of undergoing false positive results are the main obstacles for the extended use of this technique.
Normally DNA probes have at least 18 nucleotides (Lomakin, 1998) due to its poor stability and low melting temperature (Tm), also requiring additional fixation and permeabilization process with enzymes or other agents.
It is important to notice that, although very robust after optimized, the development of PNA FISH methods is, just as the development of PCR methods, extremely demanding and requires a great knowledge of the chemical and physical characteristics of the different parameters involved.
Furthermore, it is well known that having a PNA FISH method working for an organism does not warrant that other sequences targeting the same organism will function.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Peptide nucleic acid probe (PNA), kit and method for detection of aspergillus fumigatus and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Distinction of Aspergillus fumigatus Strains from Other Filamentous Fungi

[0111]PNA Probe Sequence:

SEQ ID No. 15′- ACA GAG CAG GTG ACA -3′(coupled to Alexa Fluor 594).

[0112]A. fumigatus and other filamentous fungi strains, capable to form conidia were maintained in Sabouraud dextrose agar or Potato dextrose agar for approximately 7 days at room temperature. For each experiment, conidia were harvested by flooding the agar surface with sterilized saline solution containing NaCl 8.00 g.L-1; KCl 0.2 g.L-1 ; Na2HPO4.2H2O 1.44 g.L-1; KH2PO4 0.24 g.L-1 (pH 7.4). Biomass was then suspended in the saline solution and collected with a pipette tip to a sterile tube. The heavier fragments were allowed to deposit in the bottom for 5-10 minutes and subsequently the supernatant was transferred to a new sterile tube.

[0113]The suspension is centrifuged (10 minutes; 10.000 g) to wash the sample. Subsequently 1×106 cells ml−1 of that suspension were resuspended in peptone-yeast extract-glucose (PYG) co...

example 2

Detection of A. fumigatus in Different Clinical Samples (Blood and Sputum)

[0123]PNA Probe Sequence:

SEQ ID No. 15′- ACA GAG CAG GTG ACA -3′(coupled to Alexa Fluor 594).

[0124]Sample Preparation:

[0125]Ten ml of defibrinated sheep blood or 1 ml of artificial sputum media were added to BACTEC™ Plus Aerobic / F Medium and incubated at 37° C., 120 rpm. After 6 hours germination (minimal germination time required to perform brighter fluorescence signal), 1 ml was recovered from each culture to perform hybridization on glass slides.

[0126]Fixation:

[0127]The fixation was performed according to the procedure described in Example 1.

[0128]Hybridization:

[0129]The hybridization was performed according to the described in Example 1 with the slightly difference of using distilled water, instead of saline solution, in the hybridization process steps, with the purpose of better disrupting cells (example: blood).

[0130]Wash:

[0131]The washing step was performed according to the procedure described in Exampl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
temperaturesaaaaaaaaaa
temperaturesaaaaaaaaaa
Login to View More

Abstract

The present invention refers to the development of a Peptide Nucleic Acid (PNA) probe for the detection and discrimination of Aspergillus fumigatus in different types of samples.PNA is a synthetic molecule analogue to DNA that, due to its physicochemical properties, allows a faster analysis with higher sensitivity than the DNA probes.These probes are combined with Fluorescence in situ hybridization (FISH), a molecular biology technique that allows the detection of Aspergillus fumigatus in diverse clinical samples, such as blood, serum, sputum, bronchoalveolar lavage fluid and biopsies.The combination of these two technologies rendered the FISH procedure faster, simpler and more efficient.The present invention also includes the development of the kit of detection and respective procedure for the Aspergillus fumigatus identification in clinical samples.

Description

FIELD OF THE INVENTION[0001]This invention relates to a process for the detection of microorganisms clinically relevant. For that purpose, a PNA probe for the detection and discrimination of Aspergillus fumigatus was developed.[0002]In addition to the probe, the present invention includes the PNA FISH procedure and its application to a kit for the detection of Aspergillus fumigatus in biological samples. Therefore it has clinical application.BACKGROUND OF THE INVENTION[0003]Aspergillus fumigatus is a saprophyte filamentous fungus that feeds on decaying organic matter and is able to form a type of spore, named conidium, which can survive in a wide range of aggressive environments. This protection strategy provides ubiquity to Aspergillus fumigatus allowing spreading through the air and colonizing new ecological niches. This fungus can colonize human respiratory mucosa. Depending on the host immune system, the inhaled conidia can lead to disease. In fact, A. fumigatus is the main caus...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C07K7/02C12Q1/68
CPCC12Q1/6895C07K7/02C12Q2525/107
Inventor CERQUEIRA, LAURA ISABEL MACIEIRAALMEIDA, CARINA MANUELA FEMANDESDE OLIVEIRA AZEVEDO, NUNO FILIPE RIBEIRO PINTODA COSTA VIEIRA, MARIA JOAO LOPES
Owner UNIVERSITY OF MINHO